Project Summary Osteoporosis is a common disease characterized by low bone mass and microarchitectural deterioration of the skeleton, resulting in bone fragility and increased risks for fractures. Osteoporosis is caused by an imbalance between bone resorption and bone formation in favor of bone resorption. Thus, effective inhibition of bone resorption has long been recognized as an important therapeutic strategy for osteoporosis. Osteoclasts, the bone resorbing cell, differentiate from cells of the monocyte/macrophage lineage upon stimulation by M-CSF and RANKL. RANKL exerts these diverse functions by binding and activating its receptor RANK. Mice lacking the gene for either RANKL or RANK develop osteopetrosis due to complete failure to form osteoclast, indicating that the RANKL/RANK system is essential for osteoclast formation. Thus, RANKL was recognized as an attractive antiresorptive drug target for osteoporosis shortly after its discovery and Amgen subsequently developed a humanized anti-RANKL antibody (denosumab) for treating postmenopausal osteoporosis. However, denosumab causes several side effects such as osteonecrosis of the jaw (ONJ), increased risk of serious infections and atypical femur fractures. In particular, ONJ and increased infection risks are likely due to the suppressing effect of denosumab on the immune system since RANKL regulates immune cell development, function and survival. Also, as a biological agent, the cost of denosumab is high and the method of delivery (injection) is not ideal. Thus, a better targeting strategy would be to use small molecules to target RANK signaling pathways that are involved in osteoclast formation but not in the immune system function. We previously identified two motifs in the RANK cytoplasmic domain that regulate osteoclast formation in vitro. To assess the role of the two RANK motifs in osteoclast formation in vivo, we have generated knockin (KI) mice bearing inactivating mutations in the two RANK motifs. Osteoclast formation is dramatically reduced in the KI mice, confirming the role of these two motifs in osteoclast formation in vivo. Importantly, inactivation of these two motifs does not affect the ability of RANK to activate the signaling pathways (NF-?B and MAPK) known to mediate immune cell development and function. Hence, we hypothesize that specifically targeting these two RANK motifs has the potential to serve as effective and selective therapeutic targets for osteoporosis. Our ultimate goal is to develop efficacious and safe small molecule drugs targeting the two RANK motifs for osteoporosis. To this end, we have developed cell-based assays for identifying compounds targeting the two RANK motifs. High throughput screens of 200,000 compounds with the cell-based assay systems followed by counter screen assays have identified several compounds that potently inhibited osteoclast formation without affecting the activation of NF-?B and MAPK pathways in vitro. This proposal seeks to carry out important proof- of-concept studies to assess the conceptual and technical feasibility of this novel therapeutic targeting strategy.